Kaposi's sarcoma (KS), a multicentric angiogenic tumor of mixed cellularity, is the leading neoplasm of patients with acquired deficiency syndrome (AIDS). Current molecular- and seroepidemiologic data demonstrate that KS-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), is the infectious cause of KS. In most cases of infection, a healthy cellular immune response likely destroys the vast majority of cells that harbor actively replicating virus. In contrast, immunosuppression can tilt this equilibrium away from this predominantly latent state to one of viral reactivation, replication and widespread dissemination. This pathogenic progression depends, in part, on high levels of KSHV production. The most remarkable pre-virion structures to appear after the initiation of viral replication are the inner capsid--the icosahedrally symmetric particles that fill the nucleus and, when fully mature, harbor the linear viral genome. In stark contrast to the successful investigations into the structure and assembly of the capsids of alpha- and betaherpesviruses, (such as herpes simplex virus 1 and cytomegalovirus, respectively), attempts to explore these aspects in the gammaherpesviruses (such as Epstein-Barr virus) have made little progress. The long-term objectives of the research we describe in the proposal are to understand KSHV structure and assembly in comparison to members of the other herpesvirus subfamilies and to place these findings into the context of KS pathogenesis. Recently, we have devised methods to purify structurally intact KSHV capsids. In this application, we propose to: 1.Purify and identify the distinct capsid species formed during lytic KSHV growth using biochemical and physical separation techniques. 2.Characterize the structural components of these capsids with biochemical, electron microscopic and mass spectrometric approaches. 3.Determine the three-dimensional structure of the capsids using computer-aided analyses of electron cryomicroscopic images. 4.Investigate KSHV capsid assembly. Understanding the details of capsid structure and assembly, an early and essential step in KSHV replication, may simultaneously identify new potential targets for therapeutic intervention.